Assessment Of Translocator Protein 18 KDa In The Activation Of Microglia And As A Potential Target For The Treatment Of Alzheimer’s Disease

Objective:Translocator protein 18 KDa(TSPO)is mainly expressed in the mitochondrial outer membrane of microglia in the central nervous system.When the brain is damaged or neuroinflammation occurs,significantly increased expression of TSPO in microglia can be detected.Furthermore,clinical imaging studies also indicate that TSPO expression significantly increases in neurodegenerative and diseases related to neuroinflammation.However,it is still unclear why TSPO is highly expressed in activated microglia.Whether TSPO is involved in microglial activation and which role does it play?Does TSPO involved in Alzheimer’s disease(AD)development by affecting the activation of microglia?All these questions still need to be explored.Methods:(1)To explore the role of TSPO in microglial activation:① We constructed TSPO stable knockdown BV2 cell line and isolated primary microglial cells from TSPO knockout mice.② LPS(1 μg·mL-1)were used to drive microglia into classically(Ml)activated state.RT-qPCR and Western blot were used to detect the expression of M1 markers:IL-6,iNOS,TNF-α,IL-1β.③ IL-4(1μg·mL-1)were used to drive microglia into alternatively(M2)activated state.RT-qPCR and Western blot were used to detect the expression of M2 markers:CD206,Argl.④ Flow cytometry were used to detect microglia phagocytosis,which were incubated with the fluorescent latex beads.(2)To explore the role of TSPO on microglial mitochondrial and metabolism:① Flow cytometry were used to detect mitochondrial membrane potential in TSPO deficiency microglia,followed by incubating with tetramethylrhodamine methyl ester(TMRM).② CCK8 measurement of cell proliferation.③ Total ATP and ROS production were measured by the ATP and ROS assay kits.④ Oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)measurement of WT and TSPO-/-microglia.(3)To explore the effects of knockout TSPO on the behavior and pathology of 5×FAD transgenic mice:①Crossing mice to obtain genotype of 5 ×FAD,TSPO-/-and 5×FAD,TSPO+/+mice.② Morris water maze experiment was used to evaluate the effect of TSPO on the learning and cognitive ability of 5×FAD transgenic mice.③Pathological experiment:Aβ levels was detected by immunohistochemistry and Western blot;inflammatory cytokine(IL-6,TNF-α,IL-1β)levels were detected by RT-qPCR;immunofluorescence detection of phagocytosis of Aβ by microglia.(4)To explore the role of TSPO agonist YL-IPA08 on AD development:①YL-IPA08 was administered to the 5×FAD transgenic mice by intragastric for two months.② Morris water maze experiment was used to evaluate the effect of YL-IPA08 on learning and cognitive ability of 5XFAD transgenic mice.③ Pathological detection:Aβ protein levels were detected by immunohistochemistry and Western blot.Protein related to APP shear levels were detected by Western blot.Results:(1)TSPO deficiency inhibits microglial inflammatory factors(IL-1β,iNOS,TNF-α,IL-6)levels induced by LPS;TSPO deficiency also inhibits microglia drive to M2 state induced by IL-4,which decrease expression of Arg1 and CD206.In addition,microglial phagocytic capacity was significantly reduced after TSPO knockdown.(2)TSPO deficiency decrease mitochondrial membrane potential and damage mitochondrial function.It appears to inhibit ATP production and the proliferation of microglia,increase mitochondrial DNA(mtDNA)outflux to the cytoplasm and ROS production.TSPO deficiency inhibit microglia oxidative phosphorylation(OXPHOS)and glycolytic.(3)Knockout of TSPO accelerates learning and memory deficits in 5×FAD transgenic mice model,as reflected by longer escape latency time through a Morris water maze.Mechanically,we found knockout of TSPO aggravates Aβ deposition,inhibits microglia phagocytosis and increases neuroinflammation in prefrontal cortex through a pathological experiment.(4)Morris water maze experiment results show that YL-IPA08 significantly shorten the escape latency and increased crossing number on the platform of 5×FAD transgenic mice.Pathologically,YL-IPA08 significantly reduced the Aβ deposition in the prefrontal cortex of 5×FAD transgenic mice.Conclusion:(1)In vitro,TSPO deficiency significantly inhibit microglial metabolism,which cause microglial activation disorders and weaken microglial phagocytosis.(2)In vivo,knockout of TSPO aggravate learning and memory deficits of 5×FAD transgenic mice due to weaken microglial phagocytosis and increase neuroinflammation.In addition,TSPO new agonist YL-IPA08 significantly reverse dementia-like behavior in 5×FAD transgenic mice by alleviates Aβ burden.Together,our results reveal a critical role of TSPO in microglial activation and AD,thus providing a new idea for AD drug development.